Serum protein analysis is useful for the diagnosis of lymphoproliferative disease. Gel electrophoresis has been the traditional method of choice for such analysis. Capillary electrophoresis (CE) has also been applied to serum protein analysis. Capillary electrophoresis encompasses a large variety of separation modes, many of which provide separations that equal or exceed the quality of separations that can be performed on slab gels. One form of capillary electrophoresis that is particularly suitable and convenient for many separations is capillary zone electrophoresis (CZE). Other forms include capillary isoelectric focusing, capillary gel electrophoresis, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electroosmotic chromatography. Separations can be performed in relatively short periods of time by using high voltages, since the small diameter and thin wall of a capillary provide efficient removal of the joule heat generated by the voltage. Detection methods for CE include UV absorbance, fluorescence, electrochemical, Raman, and mass spectrometric.
In gel electrophoresis, the stained gel generally contains a pattern consisting of a series of dark bands on a light background. This gel response can be scanned to find the absorbance at a series of x positions on the gel, creating a potentially quantifiable response. This resulting response is called a densitometer trace.
A response with an x-axis of migration time is typically the data obtained from the capillary electrophoresis experiment. The migration time axis can be changed to normalized mobility by: 1) taking the reciprocal of migration time, 2) multiplying by an appropriate constant, 3) zero correcting by subtracting the electroosmotic velocity, 4) dividing by the zero corrected mobility of a charged marker, and 5) multiplying by a constant, preferably -1. This process gives a more stable x-axis, normalized mobility, allowing far more precise identification and quantitation of the electropherogram components. The appearance of this normalized data set also more closely resembles that of the analogous gel electrophoresis densitometer trace, a shape that is familiar to clinicians.
The gel densitometric or capillary electrophoretic response obtained from a normal human serum proteins sample is a series of relatively broad bands, due to the heterogeneity of the proteins species. Paraproteins, which are homogeneous, will typically be located in a very narrow, dense band on the gel. This narrow band is referred to as a "zone of restricted mobility." On the densitometric scan or in a CE experiment, a paraprotein will show up as a sharp peak in the midst of a broader response, while in the stained gel, the paraprotein will show up as a narrow dark band within a broader one. Visual inspection of the gel electrophoretic response pattern is a very subjective, technique-dependent, and labor-intensive method to detect paraproteins, but has typically been the method of choice for gel electrophoresis. The densitometric trace may also be examined for paraproteins, but this method has proven less sensitive than direct visual inspection of the gel.
Monitoring paraprotein production is necessary to detect and treat diseases, such as multiple myeloma. An individual suffering from multiple myeloma will produce one or more abnormal immunoglobulins in large amounts. The abnormal protein is a paraprotein which, if detected at an early stage, allows an aggressive treatment plan to be employed. Left undetected, a more extreme therapy may be required. Thus, it is important to properly detect paraproteins at as low a level as possible. Large levels are easily detected with the naked eye on either a slab gel or a capillary electrophoresis electropherogram, but low level production of paraproteins are not as easily detected. An automated analysis for paraproteins in capillary electrophoresis has increased sensitivity over the densitometric response, while requiring less time and expertise than direct inspection of gels.
Prior to the discoveries underlying the present invention, methods of paraprotein analysis have simply indicated the presence of paraproteins as a plus/minus. A method that also reports suspected position(s) of paraproteins will allow easier detection, identification, and monitoring of the levels of paraprotein production in an individual.